Method to Assay Coenzzyme Q10 in Blood Plasma or Blood Serum

ABSTRACT

A method is described for determining CoQ 10  concentrations in plasma samples. CoQ 10  in the plasma sample is oxidized by treating the sample with an oxidizing agent having a redox potential higher than the redox potential of COQ 10 , such as, for example, para-benzoquinone. Following oxidation of the COQ 10 , the CoQ 10  in the plasma sample is extracted with an alcohol, such as, for example, 1-propanol. The alcohol extract is analyzed using direct injection into the HPLC apparatus. This method achieves a rapid, accurate analysis of plasma CoQ 10  levels, which can be used for monitoring the bioavailability of orally administered CoQ 10  used as a food supplement or as an adjunctive therapy.

This application claims priority to U.S. patent application Ser. No. 10/444,535 filed May 23, 2003; which claims benefit to U.S. Provisional Application Ser. No. 60/382,943 filed on May 23, 2002, all of which are expressly incorporated by reference in their entireties as part of the present disclosure.

BACKGROUND OF THE INVENTION

Coenzyme Q₁₀ (2,3 dimethyl-5-methyl-6-decaprenyl benzoquinone) (“CoQ₁₀”) levels in whole blood and plasma have been the subject of much inquiry as described, for example, in Tomasetti, M., Alleva, R., Solenghi, M. D., Littarru, G. P., Distribution of antioxidants among blood components and lipoproteins: significance of lipids/CoQ₁₀ ratio as a possible marker of increased risk for atherosclerosis. BioFactors, 9, 231-240 (1999), the entire content of which is incorporated herein by reference. It is likely that plasma concentrations of CoQ₁₀ reflect an overall metabolic demand, as discussed in Littarru, G. P., Lippa, S., Oradei, A., Fiorini, R. M., Mazzanti, L., Metabolic and diagnostic implications of human blood CoQ₁₀ levels, in Biomedical and Clinical Aspects of Coenzyme Q vol. VI, (eds. K. Folkers, G. P. Littarru, T. Yamagami), Elsevier North Holland, pp. 167-178 (1991), the entire content of which is incorporated herein by reference. In addition, together with other lipophilic antioxidants, CoQ₁₀ plays an intrinsic role in protecting circulating lipoproteins against oxidative damage. Therefore, the concentration of CoQ₁₀ in lipoproteins and blood plasma could be of clinical importance regarding oxidative stress and antioxidant defense. Increased levels of CoQ₁₀ enhance its antioxidant protection, even though the potential to act as an antioxidant in vivo probably depends not only on total CoQ₁₀ concentration, but also on its redox status. The content of CoQ₁₀ in single classes of lipoproteins has been found to be strictly correlated with CoQ₁₀ plasma concentration. Previous reports have shown that the LDL-cholesterol/CoQ₁₀ ratio significantly correlates with the total-cholesterol/HDL-cholesterol ratio which is usually considered a risk factor for atherosclerosis as described, for example in Alleva, R., Tomasetti, M., Bompadre, S., Littarru, G. P., Oxidation of LDL and their subfractions: kinetic aspects and CoQ₁₀ content. Molec Asp Med, 18, s105-s112 (1997), the entire content of which is incorporated herein by reference. Some effective hypocholesterolemic agents, namely the statins, also lower plasma CoQ₁₀ concentrations, owing to the common biosynthetic pathway of cholesterol and the isoprenoide side chain of coenzyme Q as described, for example, in Mortensen, S. A., Leth, A., Agner, E., Rohde, M., Dose-related decrease of serum coenzyme Q₁₀ during treatment with HMO-CoA reductase inhibitors. Molec Asp Med, 18, s137-s144 (1997), the entire content of which is incorporated herein by reference. Therefore, it would be desirable to have an effective, reliable, fast method to measure CoQ₁₀ concentrations in blood plasma or blood serum to monitor the CoQ₁₀ levels in patients receiving hypocholesterolemic agents.

CoQ₁₀ is used as a food supplement or as an adjunctive therapy in several diseases and the blood plasma or blood serum levels achieved upon oral administration of CoQ10 can correlate with clinical efficacy. Tests of blood plasma or blood serum levels of CoQ₁₀ are useful for monitoring the bioavailability of orally administered coenzyme Q₁₀.

Several methods have been described for assaying either total CoQ₁₀ or the reduced (ubiquinol-10, CoQ₁₀H₂) and oxidized (ubiquinone-10) forms in blood plasma, and several of these methods are described in the following references, the entire contents of each of which are incorporated herein by reference: Lang, J. K, Packer, L., Quantitative determination of vitamin E and oxidized and reduced coenzyme Q by high-performance liquid chromatography with in-line ultraviolet and electrochemical detection, J Chromatogr, 385, 109-117 (1987); Finckh, B., Kontush, A., Commentz, J., Hubner, C., Burdeleski, M., Kohlschutter, A., High-performance liquid chromatography-coulometric electrochemical detection of ubiquinol 10, ubiquinone 10, carotenoids and tocopherols in neonatal plasma, in Methods in Enzymology, Vol 299, (Lester Packer Ed.), pp. 341-348, Academic Press, San Diego (1999); Podda, M., Weber, C., Traber, M. G., Milbradt, R., Packer, L., Sensitive high-performance liquid chromatography techniques for simultaneous determination of tocopherols, tocotrienols, ubiquinols and ubiquinones in biological samples in Methods in Enzymology, Vol 299, (Lester Packer Ed.), pp. 330-341, Academic Press, San Diego (1999); Finckh, B., Kontush, A., Commentz, J., Hubner, C., Burdeleski, M., Kohlschutter, A. Monitoring of ubiquinol 10, ubiquinone 10, carotenoids and tocopherols in neonatal plasma microsamples using high-performance liquid chromatography with coulometric electrochemical detection. Anal Biochem, 232, 210-216 (1985); Okamoto, T., Fukunaga, Y., Ida, Y., Kishi, T., Determination of reduced and total ubiquinones in biological materials by liquid chromatography with electrochemical detection, J Chromatogr, 430, 11-19 (1988); Grossi, G., Bargossi, A. M., Fiorella, P. L., Piazzi, S. Improved high-performance liquid chromatographic method for the determination of coenzyme Q₁₀ in plasma. J. Chromatogr., 593, 217-226 (1988); Edlund, P. O., Determination of coenzyme COQ₁₀, α-tocopherol and cholesterol in biological samples by coupled-column liquid chromatography with coulometric and ultraviolet detection. J. Chromatogr, 425, 87-97 (1988); Lagendijk, J., Ubbink, J. B., Vermaak, W. J., Measurement of the ratio between the reduced and oxidized forms of coenzyme Q10 in human plasma as a possible marker of oxidative stress, J. Lipid Res, 37, 67-75 (1996); Yamashita, S., Yamamoto, Y., Simultaneous detection of ubiquinol and ubiquinone in human plasma as a marker of oxidative stress, Anal. Biochem, 250, 66-73 (1997). As used herein, ubiquinol means reduced CoQ₁₀ and ubiquinone means oxidized CoQ₁₀.

The previous methods of analyzing blood plasma to assay the concentration of CoQ₁₀ have several disadvantages. Prior methods require that the CoQ₁₀ be extracted from the plasma, followed by drying, which concentrates the extract. Losses in CoQ₁₀ can occur during the drying and concentration step. In addition, these methods analyze the oxidized form of COQ₁₀, while most of the CoQ₁₀ in the plasma is in the reduced form. These methods rely on oxidation of the CoQ₁₀ during the extraction procedure. UV methods for assaying CoQ₁₀ usually quantify the oxidized coenzyme at 275 nm. It is commonly assumed that CoQ₁₀ is completely oxidized during the extraction and HPLC procedure, but this is not necessarily the case when the sample is fresh and the reduced form of CoQ₁₀ largely predominates. Accordingly, these methods can result in underestimates of CoQ₁₀ concentration if all of the CoQ₁₀ is not oxidized.

The present invention is directed to a new, simplified method for evaluating total CoQ₁₀ in blood plasma or blood serum. The method of the present invention results in reduced time and cost for the analysis of CoQ₁₀ in plasma or serum as compared to prior methods, and provides more accurate results.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a typical chromatogram of a plasma sample, before and after spiking with a known amount of standard. A typical chromatogram of the standard alone is also shown.

FIG. 1B shows a diode array analysis of the peak of a standard of CoQ₁₀ (50 ng).

FIG. 1C shows a diode array analysis of the peak obtained on a plasma sample analyzed by the method of the present invention.

FIG. 2 shows a linear correlation between the results obtained by analysis of a sample for CoQ₁₀ using a reference electrochemical detection method and the method of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method to assay the concentration of CoQ₁₀ in blood plasma or blood serum. The sample of blood plasma or blood serum is treated with an oxidizing agent having a redox potential higher than the redox potential of COQ₁₀, followed by extraction with an alcohol and direct injection of the alcohol extract into a High Performance Liquid Chromatography (“HPLC”) apparatus.

In a preferred embodiment of the method, the CoQ₁₀ in the plasma or serum sample is oxidized by adding para-benzoquinone as an oxidizing agent. Extraction is performed using an alcohol, preferably n-propanol. The extract is then assayed by direct injection of the propanol extract into the HPLC apparatus without bringing the extract to dryness. The method can be conducted on fresh plasma or serum samples since CoQ₁₀ present in the sample is oxidized prior to propanol extraction.

The method may be performed using an oxidizing agent other than benzoquinone. For example, other oxidizing agents having a redox potential higher than the ubiquinone/ubiquinol couple may be used in the method. Also, the extraction of the oxidized CoQ₁₀ in the plasma or serum sample may be performed using any appropriate alcohol known to those skilled in the art. For example, 1-propanol, butanol or pentanol may be used for the extraction of the oxidized CoQ₁₀.

A particularly preferred embodiment of the present method is set forth in the Example below. It should be understood that the description set forth below is not intended to limit the invention in any way, and those skilled in the art will readily understand that modifications to the reagents, equipment or other parameters set forth below can be made without departing from the spirit or scope of the invention. Results obtained using the method on various samples are also described below.

EXAMPLE Materials and Methods

Reagents

R.S. type methanol and n-propanol were used (obtained from Carlo Erba, Rodano, Milan, Italy). Ethanol was R.S. plus grade (obtained from Carlo Erba, Rodano, Milan, Italy). Benzoquinone was obtained from Sigma (St Louis, Mo., USA). Lithium perchlorate was obtained from Aldrich (Steinheim, Germany).

Solutions for the ECD (electrochemical detection) chosen as a reference method were filtered through a Nylon 66 membrane, 0.2 μm×47 mm (Supelco, Bellafonte, Pa., USA) and degassed. Pure Coenzyme Q₁₀ standard was obtained from Kaneka (Osaka, Japan). Standard solutions were in ethanol.

Samples

Blood was drawn from the cubital vein of laboratory staff, after informed consent, and anti-coagulated with lithium heparin. Plasma obtained after centrifugation at 4,000 g for 15 min., at 4° C. was used fresh, or after storage at −80° C. for the day-to-day precision assay.

In order to check the stability of total CoQ₁₀ in plasma, aliquots of 3 different samples were kept for 3 days at 4° C., room temperature (22° C.) and at −20° C. respectively.

High Performance Liquid Chromatography

The HPLC apparatus consisted of a Beckman System pump model 126, a detector model 166 (Beckman Instruments, San Ramon, Calif., USA) and an injector equipped with a 200 μl loop (Rheodyne 7725i obtained from Supelco, Milano, Italy). The column was a Supelcosil LC 18 (obtained from Supelco, Milano, Italy) 25 cm×0.46 cm i.d. 5 μ, precolumn LC 18S, 2 cm. (obtained from Supelco, Milano, Italy). An in line filter A-701 (obtained from Upchurch Scientific, Inc., Oak Harbor, Wash., USA) was placed between the injector and the precolumn. The photodiode array detector for the UV spectrum analysis of the CoQ₁₀ peak was a SPD-M (Shimadzu, Tokyo).

Description of the Analysis

200 μl of a blood plasma sample were supplemented with 50 μl of a 1,4 benzoquinone solution (2 mg/ml) in a test tube and vortexed for 10 seconds. After 10 minutes, 1 ml of n-propanol was added. The test tube was vortexed for 10 seconds and centrifuged at 10,000 rpm for 2 minutes in order to spin down the protein precipitate. 200 μl of the supernatant were injected into the HPLC. The supernatant, placed in a capped test tube, was stable for up to three days when kept at 22° C. Mobile phase was constituted by ethanol-methanol (65%-35%) and the flux was 1 ml/min. UV detection was performed at 275 nm.

200 μl of different concentrations of pure oxidized CoQ₁₀ were injected as standards. Working solutions of the standards were in propanol:water (5:1), i.e. the same propanol:water ratio as for the samples. Peak area analysis was performed by a Beckman Gold Data System (DOS version).

Coulometric Analysis

Coulometric analysis of CoQ₁₀ was performed according to a standard procedure described in Alleva, R., Tomasetti, M., Bompadre, S., Littarru, G. P., Oxidation of LDL and their subfractions: kinetic aspects and CoQ₁₀ content. Molec Asp Med. 18, s105-s112 (1997). Ubiquinol/ubiquinone separation was performed on an ODS reversed phase column (Supelcosil LC18, 15×4.6 mm i.d. 3 μm, Supelco, Milano, Italy) using a mobile phase constituted by 50 mM sodium perchlorate in methanol:ethanol (80:20), at flow rate of 1 ml/min. A Coulochem II, model 5200 electrochemical detector (ESA, Bedford, Mass., USA), with the analytical cell set at −0.5 V and +0.35 V was used to detect the oxidized and reduced forms of CoQ₁₀.

Recovery, Accuracy and Precision

Recovery of CoQ₁₀ was based on a comparison between the peaks obtained by spiking samples with increasing concentrations of oxidized CoQ₁₀ and the corresponding peaks of the standard. Recovery was documented at 3 concentrations (1.16, 2.32, and 3.48 μM) with triplicate determinations for each concentration. Intra-assay accuracy and precision were determined using 4 samples, the value of which had been certified by a reference ECD method; each level was assayed 5 times for the intra-assay accuracy and precision test. Inter-assay accuracy and precision were determined over a two-month period using a quality control sample (n=21).

Results

Chromatography and Recovery

Typical, representative chromatograms of both a standard and a plasma sample are shown in FIG. 1A. Diode array analysis of the peak with the same retention time as the CoQ₁₀ standard is also shown in FIGS. 1B and 1C. Spiking of a sample containing an initial concentration of 0.29 μM with 1.16, 2.32 or 3.48 μM CoQ₁₀ yield a recovery of 96.3, 98.1 and 98.5% as shown in Table 1. TABLE 1 Recovery of exogenous CoQ₁₀ added to a plasma sample Measured (μM) Recovery Basal concentration 0.29 Spiking with CoQ₁₀ 1.16 μM 1.40 96.3% Spiking with CoQ₁₀ 2.32 μM 2.56 98.1% Spiking with CoQ₁₀ 3.48 μM 3.72 98.5%

Calibration Curves

Calibration curves constructed using propanol/water solutions of pure CoQ₁₀ as described in the Materials and Methods section above showed linearity over a concentration range of 7.9-579 nM, corresponding to a concentration of 47.4-3474 nM CoQ₁₀ in plasma. Correlation coefficients (r²) for 20 calibration curves obtained over a two month period ranged from 0.98 to 0.999 The limit of quantitation was 0.037 μM (1.23 nmoles in column) with a precision of 10.52%.

Accuracy and Precision

Within run (intra-assay) precision (CV%) and accuracy, determined as deviation from nominal values, appear in Table 2. Four samples with different, decreasing concentrations of total CoQ₁₀, having a nominal value previously determined by the electrochemical detection method chosen as reference, were analysed five times each. As shown in Table 2, CV% becomes consistently higher than 5 only for samples less than 0.06 μM. Normal plasma values of CoQ₁₀ typically range between 0.75 and 0.98 μM. Deviation from nominal values was never higher than 4.7%. TABLE 2 Within run accuracy and precision Expected Measured concentration concentration (nM) (nM) Deviation % C.V. % 894 911 1.9% 1.6% 447 432 −3.4% 5.3% 178 170 −4.7% 5.7% 36 37 2.6% 10.5%

Day-to-day precision, conducted over a two month period, is summarized in Table 3. For values corresponding to normal plasma concentrations, accuracy and precision were comparable to that obtained in the intra-assay conditions. TABLE 3 Day to day accuracy and precision test Nominal concentration of sample 0.81 (μM) Mean of 21 measurements (μM) 0.80 % deviation from nominal value 0.7 C.V. % 2.16

Comparison with the ECD Method

Ten samples were analysed, in parallel, both by the electrochemical method (ECD) and by the method of the present invention (UV), and the results are shown in Table 4. These samples were chosen on the basis of their different levels of total CoQ₁₀ and different extent of reduction. Correlation between the results of total CoQ₁₀ obtained by the two methods appears in FIG. 2. TABLE 4 Results from a group of samples analysed by electrochemical (ECD) and by UV detection ECD Detection UV Detection Oxidized Reduced Total CoQ₁₀ Total CoQ₁₀ Sample CoQ₁₀ (μM) CoQ₁₀ (μM) (μM) Red/Tot (%) (μM) % Variation A 0.95 1.62 2.57 63.09% 2.84 10.27% B 1.47 0.30 1.78 17.12% 1.75 −1.44% C 0.02 0.74 0.76 97.36% 0.83 9.44% D 2.01 0.10 2.11 4.80% 2.18 3.50% E 0.30 1.02 1.32 77.56% 1.22 −7.51% F 0.45 0.05 0.51 10.76% 0.48 −6.62% G 2.11 0.40 2.51 15.92% 2.37 −5.53% H 0.15 1.69 1.84 92.03% 2.00 8.89% I 0.58 0.49 1.07 45.91% 1.03 −3.38% L 0.94 0.84 1.78 47.03% 1.79 0.71%

Stability

The results obtained on 3 different samples stored for 1, 2 or 3 days at different temperatures are shown in Table 5. Total CoQ₁₀ was practically stable for at least three days even when kept at room temperature. TABLE 5 Stability of CoQ₁₀ values at different storage conditions and times Time after blood withdrawal (hours) Storage 1 h. 24 h. 48 h. 72 h. Sam- condi- Mean St. Mean St. Mean St. Mean St. ple tions μM Dev. μM Dev. μM Dev. μM Dev. A Control 1.73 ± 0.05 Room 1.91 ± 0.03 1.81 ± 0.00 1.93 ± 0.03 Temp. 0-4° C. 1.97 ± 0.03 1.82 ± 0.11 1.82 ± 0.16 −20° C. 1.84 ± 0.05 1.91 ± 0.08 1.97 ± 0.05 B Control 1.06 ± 0.03 Room 1.11 ± 0.04 1.05 ± 0.02 1.02 ± 0.03 Temp. 0-4° C. 1.06 ± 0.05 1.03 ± 0.08 1.11 ± 0.03 −20° C. 1.14 ± 0.06 1.00 ± 0.00 1.09 ± 0.03 C Control 2.68 ± 0.11 Room 2.72 ± 0.12 2.80 ± 0.11 2.83 ± 0.09 Temp. 0-4° C. 2.81 ± 0.15 2.78 ± 0.01 2.94 ± 0.07 −20° C. 2.84 ± 0.11 2.80 ± 0.08 2.93 ± 0.08

Discussion of Results

Prior methods for evaluating CoQ₁₀ in plasma and biological tissues are based on alcohol-hexane extraction; the extract is brought to dryness and injected into the HPLC apparatus, where CoQ₁₀ is usually revealed and quantified at 275 nm or by electrochemical detection. An internal standard, such as CoQ₈ or CoQ₉, is often added to the sample before extraction, in order to quantify a recovery. In all previously described methods, the organic solvent extract is brought to dryness and therefore concentrated before injection into the HPLC.

In the present method the sample is only diluted with propanol and then injected into the HPLC. Direct injection of the propanol extract also makes the procedure particularly simple and fast. Prior oxidation of reduced CoQ₁₀ with para-benzoquinone eliminates the possibility of underestimating total CoQ₁₀ in fresh samples. In fact fresh samples contain almost exclusively reduced CoQ₁₀ and the usual extraction procedures are often not sufficient to completely oxidize ubiquinol. On the other hand LTV detection at 275 only reveals ubiquinone. The chromatographic features and the diode array analysis of the peak show a selective separation of CoQ₁₀, with no superimposed peaks. Reproducibility of the method is shown by a CV below 2% for samples having normal values of plasma CoQ₁₀ and about 5% for samples having a quarter of the normal amount.

The rather low retention time for CoQ₁₀, under the chromatographic conditions makes the analysis fast enough and the peak does not overlap with any other components, as shown by similar values obtained using the ECD method and by the diode array analysis of the peak.

The efficiency of CoQ₁₀ extraction by the method of the present invention appears to be very satisfactory as the addition of 1, 2 or 3 μg of CoQ₁₀ to a sample leads to a 96.3-98.5% quantitative recovery (see Table 4) which makes the use of an internal standard unnecessary. This is in agreement with data from Edlund's work (Edlund, P. O., Determination of coenzyme CoQ₁₀, α-tocopherol and cholesterol in biological samples by coupled-column liquid chromatography with coulometric and ultraviolet detection. J. Chromatogr, 425, 87-97 (1988)) where it appears that a dilution of the sample with n-propanol (1:4) leads to a 100% recovery.

Correlation with the reference ECD method gave an r=0.98 (p<0.0001) as shown in Table 4. The data of Table 4 also show the efficiency of the method in properly quantifying different concentrations of CoQ₁₀ and different extents of reduction. Fresh samples have their CoQ₁₀ almost completely reduced, see Lagendijk, J., Ubbiik, J. B., Vermaak, W. J., Measurement of the ratio between the reduced and oxidized forms of coenzyme Q10 in human plasma as a possible marker of oxidative stress, J. Lipid Res, 37, 67-75 (1996); Yamashita, S., Yamamoto, Y., Simultaneous detection of ubiquinol and ubiquinone in human plasma as a marker of oxidative stress, Anal. Biochem, 250, 66-73 (1997). Therefore, the testing described above also included some samples which had been frozen and thawed several times, where CoQ₁₀ was partially or almost completely oxidised. It is evident that the extent of reduction of CoQ₁₀ in the sample does not affect the final result.

For practical reasons the propanol extract can be kept in the refrigerator up to 3 days before injection into the HPLC. Several samples were checked by analysing them 1 hour, 1, 2 and 4 days after drawing the blood, keeping the samples at −20° C., 0-4° C., or +22° C. (room temperature) and the results, as far as total CoQ₁₀ was concerned, were the same for the same sample whether kept at different temperatures or for different lengths of time (presumably only the reduced/oxidised CoQ₁₀ ratio changed). There was no need to add any stabilizing agent to the sample.

The data reported above demonstrates that the method of the present invention is simple and reliable, it minimizes sample handling, allows a quantitative recovery of CoQ₁₀ and it does not need an internal standard. Data not reported show that it is adaptable to semi-automated procedures and can be applied to tissue homogenates.

As will be recognized by those of ordinary skill in the art based on the teachings herein, numerous changes and modifications may be made to the above-described methods without departing from its spirit or scope as defined in the appended claims. Accordingly, this detailed description of preferred embodiments is to be taken in an illustrative, as opposed to a limiting sense. 

1. A method for determining the total concentration of CoQ₁₀ in blood plasma or blood serum, comprising the steps of: (a) obtaining a sample of blood plasma or blood serum from a human; (b) treating the sample with an oxidizing agent having a redox potential than the redox potential of the ubiquinone/ubiquinol couple (c) extracting the ubiquinone-10 from the sample using an alcohol; and (d) following extraction of the ubiquinone-10, analyzing the alcohol extract using High Pressure Liquid Chromatography to determine the concentration of of ubiquinone-10, in the alcohol extract.
 2. (canceled)
 3. The method of claim 1, wherein the alcohol is selected from the group consisting of n-propanol, butanol and pentanol.
 4. The method of claim 1, wherein the alcohol is 1-propanol.
 5. (canceled)
 6. A method for determining the total concentration of CoQ₁₀ in blood plasma or blood serum, comprising the steps of: (a) obtaining a sample of blood plasma or blood serum from a human; (b) depositing an aliquot of the sample in a container; (c) adding to the sample in the container a sufficient volume of an oxidizing agent having a redox potential higher than the redox potential of ubiquinone/ubiquinol couple to oxidize ubiquinol-10 in the sample; (d) vortexing the sample containing the oxidizing agent; (e) allowing the vortexed sample containing the oxidizing agent to stand for a sufficient period of time for the oxidizing agent to oxidize ubiquinol-10 contained in the sample; (f) adding an alcohol to the sample containing the oxidizing agent to extract ubiquinone-10 from the sample; (g) vortexing the sample containing the alcohol; (b) centrifuging the sample containing the alcohol to spin down the protein precipitate; (i) removing an aliquot of the supernatant from the centrifuged sample and analyzing the aliquot of supernatant using High Pressure Liquid Chromatography to determine the concentration of ubiquinone-10 in the sample.
 7. (canceled)
 8. The method of claim 6, wherein the alcohol is selected from the group consisting of n-propanol, butanol and pentanol.
 9. The method of claim 6, wherein the alcohol is 1-propanol.
 10. (canceled)
 11. (canceled) 